Gossypium hirsutum tissue-specific promoters and their use

ABSTRACT

The present invention relates to an isolated DNA molecule selected from the group: a promoter-effective DNA molecule of  Gossypium  which is operable in embryonic seed tissues and a promoter-effective DNA molecule of  Gossypium  which is operable in chlorophyllous tissues. Use of the promoter-effective DNA molecules in chimeric genes, and preparation of expression systems, host cells, transgenic plants, and transgenic plant seeds containing such chimeric gene is also disclosed. Methods of expressing a heterologous mRNA molecule or protein or polypeptide in chlorophyllous tissue of plants or embryonic seed tissues are also disclosed.

This application claims the priority benefit of U.S. Provisional PatentApplication Ser. No. 60/223,496, filed Aug. 7, 2000, which is herebyincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to two tissue-specific promoters of cotton(Gossypium hirsutum) plants, their use in the assembly of DNA constructswhich include a heterologous coding sequence under their control, andtransgenic plants containing the DNA constructs.

BACKGROUND OF THE INVENTION

Genetic engineering of plants, which entails the isolation andmanipulation of genetic material (usually in the form of DNA or RNA),and the subsequent introduction of that genetic material into plants orplant cells, offers considerable promise to modem agriculture and plantbreeding. Increased crop values, higher yields, feed value, reducedproduction costs, pest resistance, stress tolerance, drought resistance,the production of pharmaceuticals, chemicals and biological moleculesare all potentially available through genetic engineering techniques.

Methods for producing transgenic plants are well known. In a typicaltransformation scheme, a plant cell or plant tissue is transformed witha DNA construct, in which a “foreign” DNA molecule that is to beexpressed in the plant cell or tissue is operably linked to a DNApromoter molecule, which will direct expression of the foreign DNA inthe host cell, and to a 3′ regulatory region of DNA that will allowproper processing of the RNA transcribed from the foreign DNA. Thechoice of foreign DNA to be expressed will be based on the trait, oreffect, desired for the transformed plant. The promoter molecule isselected so that the foreign DNA is expressed in the desired plant.Promoters are regulatory sequences that determine the time and place ofgene expression. Transcription of DNA is dependent upon the presence ofa promoter which is a DNA sequence that directs the binding of RNApolymerase and thereby promotes mRNA synthesis.

Currently, the most widely used promoter for expression of foreign geneconstructs in dicot plants is the cauliflower mosaic virus (“CaMV”) 35Spromoter (Ow et al., “Transient and Stable Expression of the FireflyLuciferase Gene in Plant Cells and Transgenic Plants,” Science234:856-859 (1986)). The CaMV 35S promoter provides strong constitutiveexpression in most dicot plants, including cotton. Other promoters thatare widely used for inducing the expression of heterologous genes intransgenic plants include the nopaline synthase (NOS) gene promoter fromAgrobacterium tumefaciens (U.S. Pat. No. 5,034,322 to Rogers et al.),the CaMV 19S promoter (U.S. Pat. No. 5,352,605 to Fraley et al.),promoters derived from any of the several actin genes, which are knownto be expressed in most plant cell types (U.S. Pat. No. 6,002,068 toPrivalle et al.), and the ubiquitin promoter, which affords heterologousgene expression in many cell types.

However, to develop transgenic cottons with specialized agronomic traitssuch as fiber quality and seed nutrition components, a larger arsenal ofconstitutive and tissue-specific promoters will be required. Thecharacteristic expression patterns provided by these promoters must beanalyzed in order to determine if they can be used to express beneficialgenes in specific target tissues or developmental stages at maximumlevels. Although such promoter tests can be conducted with transientexpression assays or in model plant systems such as transgenic tobaccoand Arabidopsis, gene expression analysis in stable transgenic cottonplants provides confirmation that these promoters can be used for thedevelopment of commercial transgenic cotton. While severalfiber-specific promoters have been identified or otherwise tested intransgenic cotton plants (John and Crow, “Gene Expression in Cotton(Gossypium hirsutum L) Fiber: Cloning of the mRNAs,” Proc. Natl. Acad.Sci. U.S.A. 89:5769-5773 (1992); Rinehart et al., “Tissue-specific andDevelopmental Regulation of Cotton Gene FbL2A,” Plant. Physiol.112(3):1331-1341 (1996); Dang et al., “Expression of a Cotton Fiber GenePromoter in Tobacco,” Proc. Beltwide Cotton Conf., San Antonio, Tex.,Jan. 4-7, 1995 (Natl. Cotton Counc. Am., Memphis, Tenn.); Song et al.,“Expression of a Promoter from a Fiber-specific Acyl Carrier ProteinGene in Transgenic Cotton Plants,” Proc. Beltwide Cotton Conf., SanDiego, Calif., Jan. 5-9, 1998 (Natl. Cotton Counc. Am., Memphis, Tenn.),other tissue-specific promoters would be desirable to afford differentexpression characteristics for foreign genes in transgenic plants.

The present invention overcomes these and other deficiencies in the art.

SUMMARY OF THE INVENTION

A first aspect of the present invention relates to an isolated DNAmolecule selected from the group of: a promoter-effective DNA moleculeof Gossypium which is operable in embryonic seed tissues; and apromoter-effective DNA molecule of Gossypium which is operable inchlorophyllous tissues.

A second aspect of the present invention relates to a chimeric geneincluding: a promoter region which includes a first DNA molecule that isa promoter region of the present invention; a coding region operablylinked 3′ to the promoter region, the coding region comprising a secondDNA molecule encoding an mRNA molecule or a protein or polypeptide; anda 3′ regulatory region operably linked 3′ of the coding region.Expression systems, host cells, and transgenic plants and plant seedswhich carry the chimeric gene of the invention are also disclosed.

A third aspect of the present invention relates to a method of making atransgenic plant including: transforming a plant cell or tissue with achimeric gene of the present invention; and regenerating a transgenicplant from the transformed plant cell or tissue.

A fourth aspect of the present invention relates to a method ofexpressing a heterologous mRNA molecule or protein or polypeptide inchlorophyllous tissue of plants, said method including: transforming aplant cell or tissue with a chimeric gene of the present invention thatcontains a promoter-effective DNA molecule of Gossypium which isoperable in chlorophyllous tissues; and regenerating a plant from thetransformed plant cell or tissue, wherein expression of the mRNAmolecule or protein or polypeptide occurs in chlorophyllous tissue ofthe plant.

A fifth aspect of the present invention relates to a method ofexpressing a heterologous mRNA molecule or protein or polypeptide inembryonic seed tissues including: providing a plant seed of the presentinvention which includes a chimeric gene containing a promoter-effectiveDNA molecule of Gossypium which is operable in embryonic seed tissues;and propagating the plant seed under conditions effective to yield atransgenic plant which expresses the mRNA molecule or the protein orpolypeptide in embryonic seed tissues.

A sixth aspect of the present invention relates to a method ofexpressing a heterologous mRNA molecule or protein or polypeptide inchlorophyllous tissues including: providing a plant seed of the presentinvention which includes a chimeric gene containing a promoter-effectiveDNA molecule of Gossypium which is operable in chlorophyllous tissues;and propagating the plant seed under conditions effective to yield atransgenic plant which expresses the mRNA molecule or the protein orpolypeptide in chlorophyllous tissues.

In the past few years, genetically modified cottons carrying insect andherbicide resistance genes have been successfully commercialized.Transgenic cottons are likely to play an increasingly important role inworldwide cotton production by conferring useful agronomic and fibertraits. The present invention identifies two promoters of Gossypium andtheir expression patterns. One directs seed-specific expression intransgenic plants (Gh-sp) and the other directs leaf-specific expressionin transgenic plants (Gh-rbcS). Based on the patterns of GUS reportergene expression under control of the promoters of the present inventionin transgenic cotton, it is believed that chimeric genes which containthese promoters accurately reflect the expression of native genes. Thus,the present invention demonstrates that promoters from native cottongenes, when introduced into a chimeric gene, can effectively mimic theexpression of endogenous genes and do not appear to be greatly affectedby gene silencing.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a map illustrating the structure of the Gh-sp::GUS construct,which was derived from the binary vector pCGN1578. The chimericβ-glucuronidase (“GUS”) reporter gene includes the Gh-sp promoterligated upstream of the GUS coding sequence and the CaMV 35S terminatorfragment ligated downstream of the GUS coding sequence. (RB=rightborder, LB=left border, GentR is the gentamycin resistance gene, andNPTII is the neomycin transferase gene.)

FIG. 2 is a map illustrating the structure of the Gh-rbcS::GUSconstruct, which was derived from the binary vector pCGN1578. Thechimeric GUS reporter gene includes the Gh-rbcS promoter ligatedupstream of the GUS coding sequence and the CaMV 35S terminator fragmentligated downstream of the GUS coding sequence. (RB=right border, LB=leftborder, GentR is the gentamycin resistance gene, and NPTII is theneomycin transferase gene.)

FIGS. 3A-B illustrate the seed-specific expression of the Gh-sp promoterof the present invention. Immature seeds 25 days post anthesis (“DPA”)from non-transformed cotton plants (control, FIG. 3A) and a transgeniccotton plant containing a reporter gene construct with a promoter from acotton seed protein gene (Gh-sp::GUS-1, FIG. 3B) were stained forβ-glucuronidase (“GUS”) activity. No detectable GUS activity was seen incontrol seeds (FIG. 3A). Embryos of seeds that contained theGh-sp::GUS-1 reporter stained intensely blue with strongest signal inthe cotyledons, indicating high levels of GUS activity in these organs(FIG. 3B).

FIG. 4 is a graph illustrating quantitative assays for GUS specificactivity in extracts of developing ovules from untransformed cottonplants (control: ⋄) and two independent transgenic cotton plants thatcontain the Gh-sp reporter gene construct (Gh-sp::GUS-1: ▪, andGh-sp::GUS-2: Δ). Background levels of GUS activity were seen in bothcontrol and transgenic plants through 20 DPA. A rapid increase in GUSactivity occurred in transgenic seeds during the next 10 days ofmaturation, reaching maximum levels at about 30 DPA.

FIG. 5 is an image illustrating segments of expanding leaves fromnon-transformed (Control) cotton plants and transgenic cotton plantsthat contained a reporter gene with a promoter from a cotton rbcS gene(Gh-rbcS::GUS-1). The expanding leaves were stained for GUS activity.While GUS activity was not detected in leaves of control plants, leavesfrom the transgenic plant stained intensely blue indicating high levelsof GUS activity.

FIG. 6 is a graph illustrating quantitative assays of GUS specificactivity in extracts from expanding leaves from untransformed plants(Control) cotton plants and five independent transgenic cotton plantscontaining the Gh-rbcS::GUS transgenic cotton plant containing a GUSreporter gene controlled by a CaMV 35S promoter (35S::GUS). Levels ofGUS activity in all of the Gh13::GUS plants were substantially higherthan in control plants but activity varied by as much as 300% betweentransgenic lines. The two highest expressing plants (Gh-rbcS::GUS-1 andGh-rbcS::GUS-2) had activities similar to those in leaves of a typical35S::GUS containing plants.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to a pair of Gossypium-derived DNA moleculeswhich include promoter-effective DNA sequences for tissue-specificexpression of native or foreign DNA molecules under their control.

A first Gossypium promoter is a DNA molecule which includes apromoter-effective DNA sequence that provides for expression of nativeor foreign DNA in chlorophyllous tissues of transgenic plants,preferably leaf tissue. According to one embodiment, the first Gossypiumpromoter is a Gossypium hirsutum promoter characterized by a nucleotidesequence according to SEQ ID No: 1 as follows:

cgctcatgtt aacaattaat tcctataatc gacatcaaaa ttatatgaaa gaattaacac  60ttggttaccg agttaccata tttgaagata aggcgaaagg taaaaacaca aaaggcaagc 120atgaccaagc aaacaaggta tggacataga ttttttttga atcgggaatg gccaaatggg 180accgtgaaga ggggacaaag gagaaatcag gcattcacgg tttccattgg atgaaatgag 240ataagatcac tgtgcttctt ccacgtggca ggttgccaaa agataaggct ttaccattca 300agaaaagttt ccaccctctt tgtggtcata atggttgtaa tgtcatctga tttaaggatc 360caacggtcac cctttctccc aaaccaatct ctaaatgttg tgaagcttag gccaaatttt 420atgactatat ataggggatt.gcaccaaggc agtgacacta ttaagggatc agtgagactc 480ttttgtataa ctgtagcata tagtactagt aagcagtaat ag 522

A second Gossypium promoter is a DNA molecule which includes apromoter-effective DNA sequence that provides for expression of nativeor foreign DNA in embryonic plant seed tissues, preferably cotyledontissues. According to one embodiment, the second Gossypium promoter is aGossypium hirsutum promoter characterized by a nucleotide sequenceaccording to SEQ ID No: 2 as follows:

tttcagaacc aggtcgatag ttgaattagt tatgttattg gtccgactag tttgattaaa   60aattattaaa aattcataaa ataagaatag aaaaatcgct ctaatcaagt tttttagttc  120gacaagtacc aattcatgga tcaacctgct taacctcttg ttttggacaa tacctcaacc  180gcttcttgat ccaatcggtt cggatcacta aaatacccct agaaggagat gaggctaagc  240agagcgaaaa taactttcca cgagacgaga atggaaacta ttgtatttaa atattttgat  300tggattcaac aatcaatatt ttgtaaaggt aagtatttcc ataaactatt taagaataat  360gattcttcat gtgcagaacg cggcggtact attttcaaga tacaatacat tactcgacgg  420aacaattcgt attgcagtac caattatttt aaactgaatg aaatttagaa acacacaaga  480aaaaaataat ataattataa aagtatcatt gtcttggaac tcagttctat attaattctc  540atttttggtg tttatatata gaatactaag aggtactgct tctttgaaaa gacacaacat  600tttccttaga aaaaattatg aatagttata tatatttacg taaagacacc tctctttaat  660tacatttttc tttctttcct attatatata ttataaataa tataaaactt taatactata  720tattttattt gaaattactt tataatatat aatataaatt atttatatgt tatatattat  780atacaacaat tattagtaag ttaagattga atcagaaaaa atattacgag tcaaatagtt  840ttttactttg ttttataata aaaaagtaat taaaataaat ttagccccaa taaaaaaaat  900taaatctact ctttaggtga aatttttaat taattagtcc ctgaggtaag ctttcggctg  960ctaagctatg aaattgtcat tatgtataac ttttatgcaa gtgtccctca cctctcggac 1020acctccctcc ttcacaaaac agcgaggtgt acgctcacgt gtcaatgttg ggttacgtgt 1080taaggctcca acattccgat ccaccggtca atcccctctg tgtactctgt gtacataagc 1140tgtgccccat atacaaacac caacggagct caacaaagta tctgtacggt accgcattat 1200atttttattg accca 1215

Fragments of the nucleotide sequence given as SEQ ID No: 1 or SEQ ID No:2 which induce expression of heterologous DNA in transgenic plants arealso suitable promoter DNA sequences for use in a chimeric gene of thepresent invention (infra). The fragments can be prepared by using PCRprimers which direct cloning of a smaller portion of the nucleotidesequence of SEQ ID Nos: 1 or 2, and then PCR cloning the desiredfragment and isolating the same. The fragment can be inserted into achimeric gene and the chimeric gene tested to determine whether thefragment is a promoter-effective region. Efficacy of such fragments canbe based on a comparison thereof with the full length SEQ ID Nos: 1 or2.

This invention also relates to a chimeric gene which includes a promoterregion including a first DNA molecule as described above; a codingregion operably linked 3′ to the promoter region, the coding regionincluding a second DNA molecule encoding an mRNA molecule or a proteinor polypeptide; and a 3′ regulatory region operably linked 3′ of thecoding region.

The second DNA molecule is preferably heterologous DNA, which can encodeany suitable heterologous RNA molecule (translatable, non-translatable,antisense, inhibitory RNA, etc.) or heterologous protein or polypeptidethat confers to the transgenic plant containing the chimeric gene (ortransgenic plant grown from a transgenic plant seed containing thechimeric gene) a desired trait. As used herein, the term “heterologousDNA” refers to a DNA segment that has been isolated or derived from onegenotype, preferably amplified and/or chemically altered, and laterintroduced into a plant that may be a different genotype. HeterologousDNA does not generally include DNA of the same genotype, but“heterologous DNA” as used herein also includes DNA of the same genotypefrom which the amplified, chemically altered, or otherwise manipulated,DNA was first derived. Modification of the heterologous DNA sequence mayoccur, for example, by treating the DNA with a restriction enzyme togenerate a DNA fragment which is capable of being operably linked to apromoter of the present invention. Modification can also occur bytechniques such as site-directed mutagenesis or via PCR using primersdesigned to introduce a particular sequence, such as a restriction site.“Heterologous DNA” also includes DNA that is completely synthetic,semi-synthetic, or biologically derived, such as DNA derived from RNA.“Heterologous DNA” also includes, but is not limited to, non-plant genessuch as those from bacteria, yeasts, animals, or viruses; modifiedgenes, portions of genes, chimeric genes, as well as DNA that encodesfor amino acids that are chemical precursors or biologics of commercialvalue, such as polymers or biopolymers. Pool et al., “In Search of thePlastic Potato,” Science 245:1187-1189 (1989), which is herebyincorporated by reference in its entirety. Suitable heterologous DNA isany DNA for which expression in the chlorophyllous or embryonic seedtissue is beneficial to the plant or for which it is otherwisebeneficial to have the DNA expressed selectively in the chlorophyllousor embryonic seed of the plant.

The heterologous RNA molecule or protein or polypeptide can be intendedto modify a particular phenotype of the plant or, alternatively, theheterologous RNA molecule or protein or polypeptide can be substantiallyinert with respect to the plant, having its activity only when the planttissue in which it is expressed is consumed by another organism.

With respect to heterologous proteins or polypeptides to be expressed inchlorophyllous tissues, suitable proteins or polypeptides encoded by thesecond DNA molecule can include, without limitation, an herbicideresistance protein or polypeptide such as phosphinothricin N-transferase(De Block, EMBO J. 6:2513 (1987), which is hereby incorporated byreference in its entirety), glyphosate resistance (EPSP synthaseprotein) (U.S. Pat. No. 4,535,060 to Comai et al., which is herebyincorporated by reference in its entirety), and chlorsulfuron resistance(Haughn et al., Mol. Gen. Genet. 211:266 (1988), which is herebyincorporated by reference in its entirety); a pest or pathogenresistance protein or polypeptide such as Bacillus thuringiensis toxin(U.S. Pat. No. 5,990,383 to Warren et al., which is hereby incorporatedby reference in its entirety), crystal proteins (U.S. Pat. No. 4,996,155to Sick et al., which is hereby incorporated by reference in itsentirety), protease inhibitors (Ryan, Annu. Rev. Phytopathol. 38:425-449(1990) and Mundy et al., Planta 169:51-63 (1986), each of which ishereby incorporated by reference in its entirety), chitinases andchitobiases (U.S. Pat. No. 5,290,687 to Suslow et al., U.S. Pat. No.5,378,821 to Harman et al., and U.S. Pat. No. 5,446,138 to Blaiseu etal., each of which is hereby incorporated by reference in its entirety),lectins (EP Patent Application No. 351,924 A to Shell, which is herebyincorporated by reference in its entirety), lytic peptides (e.g.,apidaceins, attacins, cecropins, caerulins, bombinins, lysozymes,magainins, melittins, sapecins, sarcotoxins, haloperoxidases, andxenopsins), and elicitors (e.g., defensins, elicitins, harpins) (U.S.Pat. No. 4,705,777 to Lehrer et al., U.S. Pat. No. 5,849,868 to Beer etal., U.S. Pat. No. 5,776,889 to Wei et al., U.S. Pat. No. 5,850,015 toBauer et al., and U.S. Pat. No. 5,708,139 to Collmer et al., each ofwhich is hereby incorporated by reference in its entirety);pathogen-derived proteins such as coat proteins and replicases (Beachyet al., Rev. Phytopathol. 28:451-474 (1990), WO 90/02184 to Gonsalves etal., U.S. Pat. No. 5,510,253 to Mitsky et al., and U.S. Pat. No.5,503,999 to Jilka et al., each of which is hereby incorporated byreference in its entirety); vaccines and antibodies (Tavladorki et al.,Nature 366:469 (1993), which is hereby incorporated by reference in itsentirety); and enzymes of any source organism. Under control of theGh-RbcS promoter, such proteins or polypeptides can be expressedselectively in the leaf, and to a lesser extent the stem, of the plant,and will not interfere with food crop or root system development.

With respect to heterologous RNA to be expressed in chlorophylloustissues, suitable RNA molecules which are encoded by the second DNAmolecule can include, without limitation, antisense delta-cadinenesynthase RNA, and antisense plant virus RNA (e.g., for coat protein,replicase, etc.).

With respect to heterologous proteins or polypeptides to be expressed inembryonic seed tissues, suitable proteins or polypeptides encoded by thesecond DNA molecule can include, without limitation, a protein whichmodifies amino acid content in seed tissues (U.S. Pat. No. 5,559,223 toFalco et al., which is hereby incorporated by reference in itsentirety), a protein which modifies fatty acid content of seed tissues,and a protein or polypeptide which modifies gossypol content. Otherapproaches include the use of plants as an alternative topetrochemicals. In that respect, the current emphasis is on increasingthe production of lipids naturally produced by plants, and the need toincrease the storage capacity of plants for useful products such asfatty acids and lipids. See U.S. Pat. No. 5,602,321 to Maliyakal, whichis hereby incorporated by reference in its entirety. In certaincircumstances, any of the above-identified proteins or polypeptidesuseful for expression in chlorophyllous tissues can also be expressed inembryonic seed tissues.

The DNA construct of the present invention also includes an operable 3′regulatory region, selected from among those which are capable ofproviding correct transcription termination and polyadenyation of mRNAfor expression in plant cells, operably linked to the a DNA moleculewhich encodes for a protein of choice. A number of 3′ regulatory regionsare known to be operable in plants. Exemplary 3′ regulatory regionsinclude, without limitation, the nopaline synthase 3′ regulatory region(Fraley, et al., “Expression of Bacterial Genes in Plant Cells,” Proc.Nat'l Acad. Sci. USA, 80:4803-4807 (1983), which is hereby incorporatedby reference in its entirety) and the cauliflower mosaic virus 3′regulatory region (Odell, et al., “Identification of DNA SequencesRequired for Activity of the Cauliflower Mosaic Virus 35S Promoter,”Nature, 313(6005):810-812 (1985), which is hereby incorporated byreference in its entirety). Virtually any 3′ regulatory region known tobe operable in plants would suffice for proper expression of the codingsequence of the DNA construct of the present invention.

The promoter region, the coding region, and the 3′ regulatory region canbe ligated together using well known molecular cloning techniques asdescribed in Sambrook et al., Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor Press, NY (1989), which is herebyincorporated by reference in its entirety.

The DNA construct can also include a DNA molecule encoding a secretionsignal. A number of suitable secretion signals are known in the art andothers are continually being identified. The secretion signal can be aDNA leader which directs secretion of the subsequently translatedprotein or polypeptide, or the secretion signal can be an amino terminalpeptide sequence that is recognized by a host plant secretory pathway.The secretion-signal encoding DNA molecule can be ligated between thepromoter and the protein-encoding DNA molecule, using known molecularcloning techniques as described in Sambrook et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor Press, N.Y.(1989), which is hereby incorporated by reference in its entirety.

A further aspect of the present invention includes an expression systemthat includes a suitable expression vector in which is inserted achimeric gene of the present invention. In preparing the chimeric genefor expression, the various DNA sequences may normally be inserted orsubstituted into a bacterial plasmid. Any convenient plasmid may beemployed, which will be characterized by having a bacterial replicationsystem, a marker which allows for selection in a bacterium and generallyone or more unique, conveniently located restriction sites. Numerousplasmids, referred to as transformation vectors, are available for planttransformation. The selection of a vector will depend on the preferredtransformation technique and target species for transformation.

A variety of vectors are available for stable transformation usingAgrobacterium tumefaciens, a soilborne bacterium that causes crown gall.Crown gall are characterized by tumors or galls that develop on thelower stem and main roots of the infected plant. These tumors are due tothe transfer and incorporation of part of the bacterium plasmid DNA intothe plant chromosomal DNA. This transfer DNA (T-DNA) is expressed alongwith the normal genes of the plant cell. The plasmid DNA, pTI, orTi-DNA, for “tumor inducing plasmid,” contains the vir genes necessaryfor movement of the T-DNA into the plant. The T-DNA carries genes thatencode proteins involved in the biosynthesis of plant regulatoryfactors, and bacterial nutrients (opines). The T-DNA is delimited by two25 bp imperfect direct repeat sequences called the “border sequences.”By removing the oncogene and opine genes, and replacing them with a geneof interest, it is possible to transfer foreign DNA into the plantwithout the formation of tumors or the multiplication of Agrobacteriumtumefaciens. Fraley, et al., “Expression of Bacterial Genes in PlantCells,” Proc. Nat'l Acad. Sci., 80: 4803-4807 (1983), which is herebyincorporated by reference in its entirety.

Further improvement of this technique led to the development of thebinary vector system. Bevan, M., “Binary Agrobacterium vectors for planttransformation,” Nucleic Acids Res. 12:8711-8721 (1984), which is herebyincorporated by reference in its entirety. In this system, all the T-DNAsequences (including the borders) are removed from the pTi, and a secondvector containing T-DNA is introduced into Agrobacterium tumefaciens.This second vector has the advantage of being replicable in E. coli aswell as A. tumefaciens, and contains a multiclonal site that facilitatesthe cloning of a transgene. An example of a commonly used vector ispBin19. Frisch, et al., “Complete sequence of the binary vector Bin19,”Plant Molec. Biol. 27:405-409 (1995), which is hereby incorporated byreference in its entirety. Any appropriate vectors now known or laterdescribed for plant transformation are suitable for use with the presentinvention.

U.S. Pat. No. 4,237,224 issued to Cohen and Boyer, which is herebyincorporated by reference in its entirety, describes the production ofexpression systems in the form of recombinant plasmids using restrictionenzyme cleavage and ligation with DNA ligase. These recombinant plasmidsare then introduced by means of transformation and replicated inunicellular cultures including procaryotic organisms and eukaryoticcells grown in tissue culture.

A further aspect of the present invention includes a host cell whichincludes a chimeric gene of the present invention. As described morefully hereinafter, the recombinant host cell can be either a bacterialcell (e.g., Agrobacterium) or a plant cell. In the case of recombinantplant cells, it is preferable that the chimeric gene is stably insertedinto the genome of the recombinant plant cell.

The chimeric gene can be incorporated into cells using conventionalrecombinant DNA technology. Generally, this involves inserting thechimeric gene into an expression vector or system to which it isheterologous (i.e., not normally present). As described above, thechimeric gene contains the necessary elements for the transcription andtranslation in plant cells of the heterologous second DNA molecule.

Once the chimeric gene of the present invention has been prepared, it isready to be incorporated into a host cell. Recombinant molecules can beintroduced into cells via transformation, particularly transduction,conjugation, mobilization, or electroporation. The DNA sequences arecloned into the vector using standard cloning procedures in the art, asdescribed by Sambrook et al., Molecular Cloning: A Laboratory Manual,Second Edition, Cold Springs Laboratory, Cold Springs Harbor, N.Y.(1989), which is hereby incorporated by reference in its entirety.Suitable host cells include, but are not limited to, bacteria, virus,yeast, mammalian cells, insect, plant, and the like. Preferably the hostcells are either a bacterial cell or a plant cell.

Accordingly, another aspect of the present invention relates to a methodof making a transgenic plant. Basically, this method is carried out bytransforming a plant cell or tissue with a chimeric gene of the presentinvention and then regenerating a transgenic plant from the transformedplant cell or tissue. Preferably, the chimeric gene of the presentinvention is stably inserted into the genome of the recombinant plantcell(s) or tissue as a result of the transformation.

One approach to transforming plant cells with a chimeric gene of thepresent invention is particle bombardment (also known as biolistictransformation) of the host cell. This can be accomplished in one ofseveral ways. The first involves propelling inert or biologically activeparticles at cells. This technique is disclosed in U.S. Pat. Nos.4,945,050, 5,036,006, and 5,100,792, all to Sanford, et al., which arehereby incorporated by reference in their entirety. Generally, thisprocedure involves propelling inert or biologically active particles atthe cells under conditions effective to penetrate the outer surface ofthe cell and to be incorporated within the interior thereof. When inertparticles are utilized, the vector can be introduced into the cell bycoating the particles with the vector containing the heterologous DNA.Alternatively, the target cell can be surrounded by the vector so thatthe vector is carried into the cell by the wake of the particle.Biologically active particles (e.g., dried bacterial cells containingthe vector and heterologous DNA) can also be propelled into plant cells.Other variations of particle bombardment, now known or hereafterdeveloped, can also be used.

Another method of introducing the chimeric gene of the present inventioninto a host cell is fusion of protoplasts with other entities, eitherminicells, cells, lysosomes, or other fusible lipid-surfaced bodies thatcontain the chimeric gene. Fraley, et al., Proc. Natl. Acad. Sci. USA,79:1859-63 (1982), which is hereby incorporated by reference in itsentirety.

The chimeric gene of the present invention may also be introduced intothe plant cells by electroporation. Fromm, et al., Proc. Natl. Acad.Sci. USA, 82:5824 (1985), which is hereby incorporated by reference inits entirety. In this technique, plant protoplasts are electroporated inthe presence of plasmids containing the DNA construct. Electricalimpulses of high field strength reversibly permeabilize biomembranesallowing the introduction of the plasmids. Electroporated plantprotoplasts reform the cell wall, divide, and regenerate.

Another method of introducing the chimeric gene into plant cells is toinfect a plant cell with Agrobacterium tumefaciens or Agrobacteriumrhizogenes previously transformed with the chimeric gene. Underappropriate conditions known in the art, the transformed plant cells ortissues are grown to form shoots or roots, and develop further intoplants. Generally, this procedure involves inoculating the plant tissuewith a suspension of bacteria and incubating the tissue for 48 to 72hours on regeneration medium without antibiotics at 25-28° C.

Agrobacterium is a representative genus of the Gram-negative familyRhizobiaceae. Its species are responsible for crown gall (A.tumefaciens) and hairy root disease (A. rhizogenes). The plant cells incrown gall tumors and hairy roots are induced to produce amino acidderivatives known as opines, which are catabolized only by the bacteria.The bacterial genes responsible for expression of opines are aconvenient source of control elements for chimeric expression cassettes.In addition, assaying for the presence of opines can be used to identifytransformed tissue.

Heterologous genetic sequences such as a DNA construct of the presentinvention can be introduced into appropriate plant cells by means of theTi plasmid of A. tumefaciens or the Ri plasmid of A. rhizogenes. The Tior Ri plasmid is transmitted to plant cells on infection byAgrobacterium and is stably integrated into the plant genome. Schell,J., Science, 237:1176-83 (1987), which is hereby incorporated byreference in its entirety.

Plant tissue suitable for transformation include, but are not limited toleaf tissue, root tissue, meristems, zygotic and somatic embryos,megaspores and anthers.

After transformation, the transformed plant cells or tissues can beselected and whole plants regenerated.

Preferably, transformed cells are first identified using a selectionmarker simultaneously introduced into the host cells along with thechimeric gene of the present invention. Suitable selection markersinclude, without limitation, markers coding for antibiotic resistance,such as the nptII gene which confers kanamycin resistance (Fraley, etal., Proc. Natl. Acad. Sci. USA, 80:4803-4807 (1983), which is herebyincorporated by reference in its entirety) and the dhfr gene, whichconfers resistance to methotrexate (Bourouis et al., EMBO J. 2:1099-1104(1983), which is hereby incorporated by reference in its entirety). Anumber of antibiotic-resistance markers are known in the art and othersare continually being identified. Any known antibiotic-resistance markercan be used to transform and select transformed host cells in accordancewith the present invention. Cells or tissues are grown on a selectionmedia containing an antibiotic, whereby generally only thosetransformants expressing the antibiotic resistance marker continue togrow. Similarly, enzymes providing for production of a compoundidentifiable by color change are useful as selection markers, such asGUS (β-glucuronidase), or luminescence, such as luciferase.

Also suitable as selection markers for the present invention are genesthat cause the overproduction of a plant product, such as thecytokinin-synthesizing ipt gene from A. tumefaciens. Localizedover-production of cytokinin can be determined by known methods, such asELISA assay. Hewelt et al., “Promoter Tagging with a Promoterless iptGene Leads to Cytokine-induced Phenotypic Variability in TransgenicTobacco Plants: Implications of Gene Dosage Effects,” Plant J. 6:879-91(1994), which is hereby incorporated by reference in its entirety. Theselection marker employed will depend on the target species; for certaintarget species, different antibiotics, herbicide, or biosynthesisselection markers are preferred.

Once a recombinant plant cell or tissue has been obtained, it ispossible to regenerate a full-grown plant therefrom.

Plant regeneration from cultured protoplasts is described in Evans, etal., Handbook of Plant Cell Cultures Vol. 1: (MacMillan Publishing Co.,New York, 1983); and Vasil I. R. (ed.), Cell Culture and Somatic CellGenetics of Plants, Acad. Press, Orlando, Vol. I, 1984, and Vol. III(1986), which are hereby incorporated by reference in their entirety.

Means for regeneration vary from species to species of plants, butgenerally a suspension of transformed protoplasts or a petri platecontaining transformed explants is first provided. Callus tissue isformed and shoots may be induced from callus and subsequently rooted.Alternatively, embryo formation can be induced in the callus tissue.These embryos germinate as natural embryos to form plants. The culturemedia will generally contain various amino acids and hormones, such asauxin and cytokinins. It is also advantageous to add glutamic acid andproline to the medium, especially for such species as corn and alfalfa.Efficient regeneration will depend on the medium, on the genotype, andon the history of the culture. If these three variables are controlled,then regeneration is usually reproducible and repeatable.

Thus, the present invention also relates to a transgenic plant whichincludes a chimeric gene of the present invention, preferably-stablytransformed into the plant genome. According to one embodiment, thetransgenic plant includes a chimeric gene of the present invention whichcontains a promoter-effective DNA molecule of Gossypium which isoperable in embryonic seed tissues. According to a second embodiment,the transgenic plants includes a chimeric gene of the present inventionwhich contains a promoter-effective DNA molecule of Gossypium which isoperable in chlorophyllous tissues. According to another embodiment, thetransgenic plant includes both type of chimeric genes of the presentinvention.

Exemplary plants of the present invention include, without limitation,all major species of rice, wheat, barley, rye, cotton, sunflower,peanut, corn, potato, sweet potato, bean, pea, chicory, lettuce, endive,cabbage, cauliflower, broccoli, turnip, radish, spinach, onion, garlic,eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber,apple, pear, melon, strawberry, grape, raspberry, pineapple, soybean,tobacco, tomato, sorghum, sugarcane, and non-fruit bearing trees such aspoplar, rubber, Paulownia, pine, and elm. It is known that practicallyall plants can be regenerated from cultured cells or tissues.

After the chimeric gene is stably incorporated in transgenic plants, itcan be transferred to other plants by sexual crossing or by preparingcultivars. With respect to sexual crossing, any of a number of standardbreeding techniques can be used depending upon the species to becrossed. Cultivars can be propagated in accord with common agriculturalprocedures known to those in the field.

Once transgenic plants of this type are produced, the plants themselvescan be cultivated in accordance with conventional procedures.

Thus, a further aspect of the present invention relates to transgenicseeds recovered from the transgenic plants. The seeds can then beplanted in the soil and cultivated using conventional procedures toproduce transgenic plants.

Recovery of the product of expression of any heterologous DNA of choiceused in the present invention will depend on the exact nature of theproduct, and the technique chosen for recovery will be known to thoseskilled in the art. Recovery of the heterologous RNA or heterologousprotein or polypeptide, if desired, will depend on the nature of thepromoter employed.

Thus, another aspect of the present invention relates to a method ofexpressing a heterologous mRNA molecule or protein or polypeptide inchlorophyllous tissue of plants. Basically, this approach is carried outby transforming a plant cell or tissue with a chimeric gene of thepresent invention which contains a promoter-effective DNA molecule ofGossypium which is operable in chlorophyllous tissues and thenregenerating a plant from the transformed plant cell or tissue, whereinexpression of the mRNA molecule or protein or polypeptide occurs inchlorophyllous tissue of the plant. Preferably, the promoter, whilepresent in other plant tissues, affords substantially no expression ofthe mRNA or protein or polypeptide in non-chlorophyllous tissues of theplant. Transformation and regeneration can be performed as describedabove.

Alternatively, a method of expressing a heterologous mRNA molecule orprotein or polypeptide in chlorophyllous tissues can be carried out byproviding a plant seed which includes a chimeric gene containing apromoter-effective DNA molecule of Gossypium which is operable inchlorophyllous tissues and then propagating the plant seed underconditions effective to yield a transgenic plant which expresses themRNA molecule or the protein or polypeptide in chlorophyllous tissues.

Still another aspect of the present invention relates to a method ofexpressing a heterologous mRNA molecule or protein or polypeptide inembryonic seed tissues. Basically, this approach is carried out byproviding a plant seed which includes a chimeric gene containing apromoter-effective DNA molecule of Gossypium which is operable inembryonic seed tissues, and then propagating the plant seed underconditions effective to yield a transgenic plant which expresses themRNA molecule or the protein or polypeptide in embryonic seed tissues.Propagating the plant seed can be carried out according to known growingprocedures.

EXAMPLES

The following examples are provided to illustrate embodiments of thepresent invention, but they are by no means intended to limit its scope.The materials and methods described below were utilized in the followingexamples.

Plant Materials

Cotton plants (Gossypium hirsutum cv. Coker 312) were used for thesource of genomic DNA and as a host for Agrobacterium-mediatedtransformation.

DNA Extraction

Genomic DNA was extracted from fully expanded leaf tissues using theprocedure of Guillement et al. (“Isolation of Plant DNA: A Fast,Inexpensive, and Reliable Method,” Plant Mol. Biol. Rep. 10(1):60-65(1992), which is hereby incorporated by reference in its entirety) andfurther purified by phenol/chloroform/isoamyl alcohol (25:24:1)extraction and ethanol precipitation.

GUS Assay

Histochemical staining and fluorometric assays were used for theanalysis of GUS expression (Jefferson, “Assaying Chimeric Genes inPlants: The GUS Gene Fusion System,” Plant Mol. Biol. Rep. 5(2):387-405(1987), which is hereby incorporated by reference in its entirety).Fresh tissues were used for the detection of GUS expression withhistochemical staining solution (0.02 M5-bromo-4-chloro-3-indolyl-β-D-glucuronide, 0.1 M NaH₂PO₄, 0.25 M EDTA.5 mM potassium ferricyanide. 5 mM potassium ferrocyanide, 1.0% (v:v)Triton X-100, pH 7.0).

For the fluorometric assay, 0.2 g fresh tissues were ground in 500 μlGUS extraction buffer (50 mM phosphate buffer, pH 7.4, 10 mMβ-mercaptoethanol, 10 mM EDTA. 0.1% sodium lauryl sarcosine, and 0.1%Triton X-100). The samples were centrifuged for 15 min at 4° C.,12,000×g. Ten microliters of supernatant was transferred in 0.5 ml GUSassay buffer (1 mM 4-methyl umbelliferyl β-D-glucuronide in extractionbuffer). After incubation at 37° C. for 2 hours, the reactions werestopped by the addition of 2 ml of 0.2 M Na₂CO₃ and the fluorescencemeasured for with a Fluorometer (Turner Model 112). Three samples weretaken from each individual plant and each sample was measured 3 times.

Example 1 Isolation of a Promoter from a CottonRibulose-1,5-Bisphosphate Carboxylase Small Unit Gene

DNA sequences of a cotton ribulose-1,5-bisphosphate carboxylase smallunit gene (Gh-rbcS) (GenBank accession number X54091, which is herebyincorporated by reference in its entirety) was identified. For PCRamplification of the 5′ region of the Gh-rbcS gene, the followingoligonucleotides were developed and used as 5′ end and 3′ end primers:

5′end primer (SEQ ID No: 3) cgctcatgtt aacaattaat tcctataatc 30 3′endprimer (SEQ ID No: 4) catcgtagta cgtgggtaag ctcgagtact 30About 1 μg of genomic DNA was used in 50 μl PCR reaction. PCR wasperformed with TaKaRA Ex Taq polymerase (Takara Shuzo Co., Ltd., Japan)under the conditions suggested by the manufacturer. PCR products wereseparated on 1.1% agarose gel and stained with ethidium bromide.

The resulting PCR product was then sequenced, identifying severaldiscrepancies with the sequence reported at Genbank Accession No.X54091, which is hereby incorporated by reference in its entirety. Thenucleotide sequence of the PCR product is as follows (SEQ ID No: 5),with the discrepancies indicated in bold typeface:

cggctcatgtt aacaattaat tcctataatc gacatcaaaa ttatatgaaa gaattaacac   60ttggttaccg agttaccata tttgaagata aggcgaaagg taaaaacaca aaaggcaagc  120atgaccaagc aaacaaggta tggacataga ttttttttga atcgggaatg gccaaatggg  180accgtgaaga ggggacaaag gagaaatcag gcattcacgg tttccattgg atgaaatgag  240ataagatcac tgtgcttctt ccacgtggca ggttgccaaa agataaggct ttaccattca  300agaaaagttt ccaccctctt tgtggtcata atggttgtaa tgtcatctga tttaaggatc  360caacggtcac cctttctccc aaaccaatct ctaaatgttg tgaagcttag gccaaatttt  420atgactatat ataggggatt gcaccaaggc agtgacacta ttaagggatc agtgagactc  480ttttgtataa ctgtagcata tagtactagt aagcagtaat agcaatggcc tcctccatga  540tctcatcggc aaccattgcc accgtgaact gctcctcccc ggcacaggcc aacatggtgg  600cccccttcac cggcctcaag tctgcctctg ctttcccagt cactaggaag gccaacaacg  660acatcacttc tcttgcaagc aatggtggga gagtgcaatg catgcaggta cttggtgatg  720cataaataca acttaaatta ccccaattgt ttgaacacaa caaattacat aaattgaatc  780aaatatatat cttggctttt gagtataggt gtggcctcct cttgggaaga agaagttcga  840gacactctca tacctccccg atcttacacc cgtacagttg gctaaggaag tagattacct  900tcttcgctct aaatggattc cttgcttgga attcgaatta gaggtgtttt cgagctctaa  960attattccat tccaacactt tattttttta gtgggatatt tgatttgatt aaatgtgttt 1020tatatgtatg tgcaggaggg attcgtgcac cgtaagtact cgagcttacc cacgtactac 1080gatg 1084

PCR was used to amplify a sub-fragment from the above PCR product thatincluded only the promoter and 5′ flanking sequences. A 5′ end primerwas modified to include a PstI site (underlined below) and the 3′ endprimer was modified to include an NcoI site at the initiation codon(underlined below) as follows:

5′end primer (SEQ ID No: 6) ctgcagcgct catgttaaca attaattcct ataatc 363′end primer (SEQ ID No: 7) gagatcatgg aggaggccat gg ctattact g 31Expected DNA fragments were cut from gels and subcloned into a TAcloning vector (pGEM-T system, Promega, Madison, Wis.). The subclonedPCR product was sequenced to verify its authenticity.

The nucleotide sequence of the subcloned PCR product is as follows (SEQID No: 8):

ctgcagcgct catgttaaca attaattcct ataatcgaca tcaaaattat atgaaagaat  60taacacttgg ttaccgagtt accatatttg aagataaggc gaaaggtaaa aacacaaaag 120gcaagcatga ccaagcaaac aaggtatgga catagatttt ttttgaatcg ggaatggcca 180aatgggaccg tgaagagggg acaaaggaga aatcaggcat tcacggtttc cattggatga 240aatgagataa gatcactgtg cttcttccac gtggcaggtt gccaaaagat aaggctttac 300cattcaagaa aagtttccac cctctttgtg gtcataatgg ttgtaatgtc atctgattta 360aggatccaac ggtcaccctt tctcccaaac caatctctaa atgttgtgaa gcttaggcca 420aattttatga ctatatatag gggattgcac caaggcagtg acactattaa gggatcagtg 480agactctttt gtataactgt agcatatagt actagtaagc agtaatagcc  atggcctcct 540ccatgatctc 550The PstI and NcoI restriction sites are underlined in SEQ ID No: 8above. The promoter subfragment, therefore, includes nt 7-528 of SEQ IDNo: 8, which is separately defined as SEQ ID No: 1.

Example 2 Isolation of a Promoter From a Cotton Seed Protein Gene

The DNA sequences of a seed protein gene (Gh-sp) (GenBank accessionnumber M19389, which is hereby incorporated by reference in itsentirety) was identified. For PCR amplification of the 5′ region of theGh-sp promoter, the following oligonucleotides were used as 5′ and 3′end primers:

5′end primer (SEQ ID No: 9) gaaccaggtc gatagttgaa ttagttatgt t 31 3′endprimer (SEQ ID No: 10) ctcagctgtt tgcatcatgg cagcatcttg 30About 1 μg of genomic DNA was used in 50 μl PCR reaction. PCR wasperformed with TaKaRA Ex Taq polymerase (Takara Shuzo Co., Ltd., Japan)under the condition suggested by the manufacturer. PCR products wereseparated on 1.1% agarose gel and stained with ethidium bromide.

The resulting PCR product was sequenced, having a nucleotide sequence asfollows (SEQ ID No: 11):

tttcagaacc aggtcgatag ttgaattagt tatgttattg gtccgactag tttgattaaa   60aattattaaa aattcataaa ataagaatag aaaaatcgct ctaatcaagt tttttagttc  120gacaagtacc aattcatgga tcaacctgct taacctcttg ttttggacaa tacctcaacc  180gcttcttgat ccaatcggtt cggatcacta aaatacccct agaaggagat gaggctaagc  240agagcgaaaa taactttcca cgagacgaga atggaaacta ttgtatttaa atattttgat  300tggattcaac aatcaatatt ttgtaaaggt aagtatttcc ataaactatt taagaataat  360gattcttcat gtgcagaacg cggcggtact attttcaaga tacaatacat tactcgacgg  420aacaattcgt attgcagtac caattatttt aaactgaatg aaatttagaa acacacaaga  460aaaaaataat ataattataa aagtatcatt gtcttggaac tcagttctat attaattctc  540atttttggtg tttatatata gaatactaag aggtactgct tctttgaaaa gacacaacat  600tttccttaga aaaaattatg aatagttata tatatttacg taaagacacc tctctttaat  660tacatttttc tttctttcct attatatata ttataaataa tataaaactt taatactata  720atacaacaat tattagtaag ttaagattga atcagaaaaa atattacgag tcaaatagtt  840ttttactttg ttttataata aaaaagtaat taaaataaat ttagccccaa taaaaaaaat  900taaatctact ctttaggtga aatttttaat taattagtcc ctgaggtaag ctttcggctg  960ctaagctatg aaattgtcat tatgtataac ttttatgcaa gtgtccctca cctctcggac 1020acctccctcc ttcacaaaac agcgaggtgt acgctcacgt gtcaatgttg ggttacgtgt 1080taaggctcca acattccgat ccaccggtca atcccctctg tgtactctgt gtacataagc 1140tgtgccccat atacaaacac caacggagct caacaaagta tctgtacggt accgcattat 1200atttttattg acccaccatg ggccagggac aacctaggag gcctcaacaa ccagcaggtc 1260aaggtgagaa ccaagagcct atcaaatatg gagatgtttt caacgtcagc ggtgagttag 1320ccaacaagcc tatcgcaccc caagatgcag ccatgatgca aacagctgag 1370

PCR was used to amplify a sub-fragment from the above PCR product thatincluded only the promoter and 5′ flanking sequences. A 5′ end primerwas modified to include a PstI site (underlined below) and the 3′ endprimer was modified to include an NcoI site at the initiation codon(underlined below) as follows:

5′end primer (SEQ ID No: 12) ctgacagtttc agaaccaggt cgatagttga 30 3′endprimer (SEQ ID No: 13) ctcctaggtt gtccctggcc catggtgggt caataaaaa 39Expected DNA fragments were cut from gels and subcloned into a TAcloning vector (pGEM-T system, Promega, Madison, Wis.). The subclonedPCR product was sequenced to verify its authenticity.

The nucleotide sequence of the subcloned PCR product is as follows (SEQID No: 14):

ctgcagtttc agaaccaggt cgatagttga attagttatg ttattggtcc gactagtttg   60attaaaaatt attaaaaatt cataaaataa gaatagaaaa atcgctctaa tcaagttttt  120tagttcgaca agtaccaatt catggatcaa cctgcttaac ctcttgtttt ggacaatacc  180tcaaccgctt cttgatccaa tcggttcgga tcactaaaat acccctagaa ggagatgagg  240ctaagcagag cgaaaataac tttccacgag acgagaatgg aaactattgt atttaaatat  300tttgattgga ttcaacaatc aatattttgt aaaggtaagt atttccataa actatttaag  360aataatgatt cttcatgtgc agaacgcggc ggtactattt tcaagataca atacattact  420cgacggaaca attcgtattg cagtaccaat tattttaaac tgaatgaaat ttagaaacac  480acaagaaaaa aataatataa ttataaaagt atcattgtct tggaactcag ttctatatta  540attctcattt ttggtgttta tatatagaat actaagaggt actgcttctt tgaaaagaca  600caacattttc cttagaaaaa attatgaata gttatatata tttacgtaaa gacacctctc  660tttaattaca tttttctttc tttcctatta tatatattat aaataatata aaactttaat  720actatatatt ttatttgaaa ttactttata atatataata taaattattt atatgttata  780tattatatac aacaattatt agtaagttaa gattgaatca gaaaaaatat tacgagtcaa  840atagtttttt actttgtttt ataataaaaa agtaattaaa ataaatttag ccccaataaa  900aaaaattaaa tctactcttt aggtgaaatt tttaattaat tagtccctga ggtaagcttt  960cggctgctaa gctatgaaat tgtcattatg tataactttt atgcaagtgt ccctcacctc 1020tcggacacct ccctccttca caaaacagcg aggtgtacgc tcacgtgtca atgttgggtt 1080acgtgttaag gctccaacat tccgatccac cggtcaatcc cctctgtgta ctctgtgtac 1140ataagctgtg ccccatatac aaacaccaac ggagctcaac aaagtatctg tacggtaccg 1200cattatattt ttattgaccc accatgggcc agggacaacc taggag 1246The PstI and NcoI restriction sites are underlined in SEQ ID No: 14above. The promoter subfragment, therefore, includes nt 7-1221 of SEQ IDNo: 14, which is separately defined as SEQ ID No: 2.

Sequence analysis of the Gh-sp promoter indicated that it contains aconserved sequence motif located 89 bp upstream of TATA box that issimilar to the G-box core motif (ACGT). Similar elements are requiredfor seed-specific gene expression in several plant species (Salberg etal., “Deletion Analysis of a 2S Seed Storage Protein Promoter ofBrassica napus in Transgenic Tobacco,” Plant Mol. Biol. 23(4):671-683(1993); Vincentz et al., “ACGT and Vicilin Core Sequence in a PromoterDomain Required for Seed-specific Expression of a 2S Storage ProteinGene are Recognized by the Opaque-2 Regulatory Protein,” Plant Mol.Biol. 34(6):879-889 (1997); Wu et al., “The GCN4 Motif in a RiceGlutelin Gene is Essential for Endosperm-specific Gene Expression and isActivated by Opaque-2 in Transgenic Rice Plants,” Plant J. 14(6):673-683(1998), which are hereby incorporated by reference in their entirety).

Example 3 Construction of Chimeric GUS Genes Using Gh-rbcS and Gh-spPromoters and Preparation of Agrobacterium Expression Vector

In order to build gene constructs including either a Gh-rbcS or a Gh-sppromoter in a GUS reporter gene, PstI sites were added to the 5′ ends ofthe promoter sequences and the initiation codons were mutated to NcoIsites. This was done by PCR amplification of the subcloned promoterfragments with primers containing the respective restriction sites andthe resulting PCR products were purified and digested with PstI andNcoI. This resulted in a 1215 bp fragment containing the Gh-sp promoterand a 522 bp fragment containing the Gh-rbcS promoter. Each of these twopromoter fragments were separately ligated into the binary vectorpCGN1578 along with a fragment containing the β-glucuronidase (GUS)reporter gene and the CaMV 35S terminator fragment. These two resultinggene cassettes, Gh-sp::GUS and Gh-rbcS::GUS, are illustrated in FIGS. 1and 2, respectively. These constructs were introduced into the disarmedAgrobacterium tumefaciens strain EHA101 for cotton transformation.

Example 4 Preparation of Transgenic Cotton Plants Expressing ChimericGUS Gene Gh-sp::GUS

Transformation of cotton plants with a Gh-sp::GUS gene construct wascarried out by inoculation of hypocotyl segments of cotton (cv. Coker312) seedlings with Agrobacterium tumefaciens and regeneration of plantsvia somatic embryogenesis as previously reported (Bayley et al.,“Engineering 2,4-D resistance into cotton,” Theor. Appl. Genet.83(3):645-662 (1992); Payton et al., “Over-expression ofChloroplast-targeted Mn Superoxide Dismutase in Cotton (Gossypiumhirsutum L., cv. Coker 312) Does Not Alter the Reduction ofPhotosynthesis After Short Exposures to Low Temperature and High LightIntensity,” Photosynthesis Res. 52:233-244 (1997), which are herebyincorporated by reference in their entirety).

Cotton plants were regenerated from two independent cell linestransformed with the Gh-sp::GUS gene construct. The presence of the GUSreporter gene in these transgenic plants was confirmed by PCRamplification. These plants were grown to flowering in a greenhouse andanalyzed for GUS expression. Different plant tissues including sectionsof emerging leaves, longitudinal section of shoots, roots, petals,mature anthers, and styles were stained for GUS activity. Afterincubation in the staining solution for 16 hours at 37° C., no bluestain was apparent in any tissues from these primary transgenic (T₀)plants indicating that the GUS gene was not expressed.

To determine the expression pattern of Gh-sp promoter in seeddevelopment, bolls from the T₀ plants were tagged and ovules wereharvested at different days post anthesis (DPA) and subsequently stainedfor GUS expression. GUS activity was not detected in developing seedsbefore 25 DPA, but very strong staining was observed in the developingseeds at 30 DPA or later (FIG. 3B). Staining was limited to embryonictissues with strongest activity in the cotyledons. Results fromquantitative fluorometric assays of GUS activity matched thehistochemical staining pattern in maturing seeds. GUS activity in ovuleextracts increased dramatically from background levels at 25 DPA (FIG.4).

The promoter sequence used in this experiment is from the lateembryogenesis abundant (Lea) class of seed protein gene (Baker et al.,“Sequence and Characterization of 6 Lea Protein and Their Genes fromCotton,” Plant Mol. Biol. 11(2):277-291 (1988), which is herebyincorporated by reference in its entirety). Generally, water loss duringlate seed formation triggers the expression of Lea genes in all higherplants that produce desiccated seeds. Study of the developmental andenvironmental induction of Lea genes in cotton indicated that most LeamRNAs started to accumulate in 30 DPA developing seeds (Hughes andGalau, “Developmental and environmental induction of Lea and LeaA mRNAsand the postabscission program during embryo culture,” Plant Cell3(6):605-618 (1991), which is hereby incorporated by reference in itsentirety). Therefore, the GUS expression pattern controlled by Gh-sppromoter in transgenic cotton plants was similar to that of theendogenous cotton Lea genes. This seed specific-promoter will be usefulin the genetic modification of seed properties such as protein quality,fatty acid composition, and gossypol content.

Example 5 Preparation of Transgenic Cotton Plants Expressing ChimericGUS Gene Gh-rbcS::GUS

Transformation of cotton plants with the Gh-rbcS::GUS gene constructswas carried out by inoculation of hypocotyl segments of cotton (cv.Coker 312) seedlings with Agrobacterium tumefaciens and regeneration ofplant via somatic embryogenesis as previously reported (Bayley et al.,“Engineering 2,4-D resistance into cotton,” Theor. Appl. Genet.83(3):645-662 (1992); Payton et al., “Over-expression ofChloroplast-targeted Mn Superoxide Dismutase in Cotton (Gossypiumhirsutum L., cv. Coker 312) Does Not Alter the Reduction ofPhotosynthesis After Short Exposures to Low Temperature and High LightIntensity,” Photosynthesis Res. 52:233-244 (1997), which are herebyincorporated by reference in their entirety).

The small subunit of ribulose-1,5-bisphosphate carboxylase (rbcS) isencoded by gene families in most plants. Promoters from one group ofthese genes contain two cis-acting elements, the I-box and the G-box,that are important for their tissue-specific expression (Donald andCashmore, “Mutation of Either G box or I box Sequences ProfoundlyAffects Expression from the Arabidopsis rbcS-1A Promoter,” EMBO J.9(3):1717-1726 (1990); Manzara et al., “Developmental and Organ-specificChanges in Promoter DNA-protein Interactions in Tomato rbcS GeneFamily,” Plant Cell 3(3):1305-1316 (1991), which are hereby incorporatedby reference in their entirety). Analysis of transgenic tomato plantsexpressing a rbcS-promoter::GUS fusion gene confirmed that promoterfragments ranging from 0.6 to 3.0 kb of rbcS1, rbcS2, and rbcS3A geneswere sufficient to confer the temporal and organ-specific expressionpattern (Manzara et al., “Developmental and Organ-specific Changes inDNA-protein Interactions in the Tomato rbcS1, rbcS2 and rbcS3 PromoterRegions,” Plant Mol. Biol. 21(1):69-88 (1993); Meier et al.,“Organ-specific Differential Regulation of a Promoter Subfamily for theRibulose-1,5-bisphosphate Carboxylase/Oxygenase Small Subunit Genes inTomato,” Plant Physiol. 107(3):1105-1118 (1995), which are herebyincorporated by reference in their entirety). In these genes, the I-boxand G-box are located within the region from −600 bp to −100 upstream oftranscription initiation site. The 522 bp promoter fragment from cottonrbcS gene that was used to develop the GUS reporter gene constructreported here does include putative I-box (nt 234-247 in SEQ ID No: 1)and G-box (nt 261-269 in SEQ ID No: 1) sequences upstream of the startcodon.

Transgenic cotton plants were regenerated from 5 independent cell linestransformed with the Gh-rbcS::GUS gene cassette and the presence of theGUS fragment was confirmed in these plants by PCR amplification ofgenomic DNA. Leaf segments from emerging leaves of plants with 4-5 trueleaves and flowering plants were incubated with GUS staining solution at37° C. overnight. GUS expression was detected in all 15 transgenicplants tested and no blue color appeared in non-transgenic plants (FIGS.5 and 6). Other tissues including roots from regenerated transgenicplantlets, longitudinal sections of shoot, petal, anthers, and 8 DPAdeveloping ovules from flowering transgenic plants were tested for GUSexpression. GUS activity was not detected in these samples except forthe shoot, which showed light staining. These results demonstrate theGUS gene expression under control of the Gh-rbcS promoter is expressedin chlorophyll-containing tissues, primarily the leaves.

Fluorometric assay of leaf tissues indicated that GUS activity in leavesof Gh-rbcS::GUS transgenic plants was significantly higher thannon-transgenic plants and transgenic plants without the GUS reportergene. There was considerable variation in the level of GUS expressionamong transgenic plants regenerated from different cell lines (FIG. 6).This is could be caused by “position effects” that depend on thechromosomal location of the transgene insertion or by co-suppressionthat is often associated with the presence of multiple transgeneinserts. Expression in transgenic cotton plants with the highest levelsof GUS activity (Gh-rbcS::GUS-1 and Gh-rbcS::GUS-2) were only slightlylower than that of a 35S::GUS transgenic plant that was also tested. Atobacco tissue-specific rbcS gene promoter has been successfully used togenerate herbicide resistant transgenic plants (Stalker et al.,“Herbicide Resistance in Transgenic Plants Expressing a BacterialDetoxification Gene,” Science 242(4877):419-423 (1988), which is herebyincorporated by reference in its entirety). Based on the GUS expressionfrom this experiment, Gh-rbcS promoter could be used to express foreigngenes at high levels in green tissues of transgenic cotton plants.

Although the invention has been described in detail for the purpose ofillustration, it is understood that such detail is solely for thatpurpose, and variations can be made therein by those skilled in the artwithout departing from the spirit and scope of the invention which isdefined by the following claims.

1. An isolated DNA molecule consisting of a seed-specificpromoter-effective DNA molecule of Gossypium which is non-constitutiveand operable predominantly in cotyledon tissue, wherein the isolated DNAmolecule consists of the nucleotide sequence of SEQ ID No:
 2. 2. Achimeric gene comprising: a promoter region comprising a first DNAmolecule according to claim 1, a heterologous coding region operablylinked 3′ to the promoter region, The heterologous coding regioncomprising a second DNA molecule encoding an mRNA molecule or a proteinor polypeptide; and a 3′ regulatory region operably linked 3′ of thecoding region.
 3. the chimeric gene according to claim 2, wherein thesecond DNA molecule encodes a protein or polypeptide selected from thegroup consisting of a protein which modifies amino acid content in seedtissues, a protein which modifies fatty acid content of seed tissues,and protein or polypeptide which modifies gossypol content.
 4. Anexpression system comprising an expression vector in which is inserted achimeric gene according to claim
 2. 5. A host cell comprising a chimericgene according to claim
 2. 6. The host cell according to claim 5,wherein the host cell is a plant cell or a bacteria cell.
 7. The hostcell according to claim 6, wherein the bacteria cell is Agrobacterium.8. The host cell according to claim 6, wherein the plant cell is a cellof a plant selected from the group consisting of rice, wheat, barley,rye, cotton, sunflower, peanut, corn, potato, sweet potato, bean, pea,chicory, lettuce, endive, cabbage, cauliflower, broccoli, turnip,radish, spinach, onion, garlic, eggplant, pepper, celery, carrot,squash, pumpkin, zucchini, cucumber, apple, pear, melon, strawberry,grape, raspberry, pineapple, soybean, tobacco, tomato, sorghum,sugarcane, poplar, rubber, Paulownia, pine, and elm.
 9. A transgenicplant comprising a chimeric gene according to claim
 2. 10. Thetransgenic plant according to claim 9, wherein the second DNA moleculeencodes a protein or polypeptide selected from the group consisting of aprotein which modifies amino acid content in seed tissues, a proteinwhich modifies fatty acid content of seed tissues, and protein orpolypeptide which modifies gossypol content.
 11. The transgenic plantaccording to claim 9, wherein the plant is selected from the groupconsisting of rice, wheat , barley, rye, cotton, sunflower, peanut,corn, potato, sweet potato, bean, pea, chicory, lettuce, endive,cabbage, cauliflower, broccoli, turnip, radish, spinach, onion, garlic,eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber,apple, pear, melon, strawberry, grape, raspberry, pineapple, soybean,tobacco, tomato, sorghum, sugarcane, poplar, rubber, Paulownia, pine,and elm.
 12. The transgenic plant according to claim 9, wherein theplant is a cotton plant.
 13. A plant seed obtained from the transgenicplant of claim 9, wherein the plant seed comprises the chimeric gene.14. the plant seed according to claim 13, wherein the transgenic plantis selected from the group consisting of rice, what, barley, rye,cotton, sunflower, peanut, corn, potato, sweet potato, bean, peachicory, lettuce, endive, cabbage, cauliflower, broccoli, turnip,radish, spinach, onion, garlic, eggplant, pepper, celery, carrot,squash, pumpkin, zucchini, cucumber, apple, pear, melon, strawberry,grape, raspberry, pineapple, soybean, tobacco, tomato, sorghum,sugarcane, poplar, rubber, Paulownia, pine, and elm.
 15. The plant seedaccording to claim 13, wherein the transgenic plant is cotton.
 16. Atransgenic plant seed comprising a chimeric gene according to claim 2.17. The transgenic plant seed according to claim 16, wherein theembryonic seed tissue is a cotyledon.
 18. The transgenic plant seedaccording to claim 16, wherein the second DNA molecule encodes a proteinor polypeptide selected from the group consisting of a protein whichmodifies amino acid content in seed tissues, a protein which modifiesfatty acid content of seed tissues, and a protein or polypeptide whichmodifies gossypol content.
 19. The transgenic plant seed according toclaim 16, wherein the transgenic plant seed is from a plant selectedfrom the group consisting of rice, wheat barley, rye, cotton, sunflower,peanut, corn, potato, sweet potato, bean, pea, chicory, lettuce, endive,cabbage, cauliflower, broccoli, turnip, radish, spinach, onion, garliceggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber,apple pear, melon, strawberry, grape, raspberry, pineapple, soybean,tobacco, tomato, sorghum, sugarcane, poplar, rubber Paulownia, pine andelm.
 20. The transgenic plant seed according to claim 16, wherein thetransgenic plant seed is from a cotton plant.
 21. a method of making atransgenic plant comprising: transforming a plant cell or tissue with achimeric gene according to claim 2, and regenerating a transgenic plantfrom the transformed plant cell or tissue.
 22. The method according toclaim 21, wherein said transforming is carried out under conditionseffective to insert the chimeric gene into the genome of the transformedplant cell or tissue.
 23. The method according to claim 21, wherein saidtransforming comprises: propelling particles at a plant cell underconditions effective for the particles to penetrate into the cellinterior and introducing an expression vector comprising the chimericgene into the plant cell interior.
 24. The method according to claim 21,wherein said transforming comprises: propelling particles at a plantcell under conditions effective for the particles to penetrate into thecell interior and introducing an expression vector comprising thechimeric gene into the plant cell interior.
 25. A method of expressing aheterologous mRNA molecule or protein or polypeptide in embryonic seedtissues comprising: providing a plant seed according to claim 16 andpropagating the plant seed under conditions effective to yield atransgenic plant which expresses the mRNA molecule or the protein orpolypeptide in embryonic seed tissues.
 26. The method according to claim25, wherein the embryonic seed tissue is a cotyledon.
 27. An isolatedDNA molecule consisting of the nucleotide sequence of SEQ ID No.
 2. 28.A chimeric gene comprising: a promoter region consisting of a first DNAmolecule according to claim 27 a coding region operably linked 3′of thecoding region.
 29. An expression system comprising an expression vectorin which is inserted a chimeric gene according to claim
 28. 30. A hostcell comprising a chimeric gene according to claim
 28. 31. A transgenicplant comprising a chimeric gene according to claim
 28. 32. A transgenicplant seed comprising a chimeric gene according to claim
 28. 33. Amethod of making a transgenic plant comprising; transforming a plantcell or tissue with a chimeric gene according to claim 28; andregenerating a transgenic plant from the transformed plant cell ortissue.
 34. A method of expressing a heterologous mRNA molecule orprotein or polypeptide in embryonic seed tissues comprising: providingplant seed according to claim 32 and propagating the plant seed underconditions effective to yield a transgenic plant which expresses themRNA molecule or the protein or polypeptide in embryonic seed tissues.35. The isolated DNA molecule according to claim 1, wherein the DNAmolecule, when present in a transgene introduced into Gossypium, iseffective to induce expression of a product encoded by the transgene inembryonic seed tissue at about 25 days post anthesis.